Methods for determining norovirus-reactive antibodies

Abstract

The present disclosure is directed to methods for determining the presence and/or amount of norovirus-reactive antibodies in a sample from a subject. The subject may be vaccinated with a norovirus vaccine or infected with a norovirus. The present disclosure further relates to in vitro methods for diagnosing a norovirus infection and determining protection against a norovirus infection in a subject for instance after vaccination with a norovirus vaccine. The present disclosure is further directed to kits for determining norovirus-reactive antibodies in a sample. The present disclosure is further directed to microsphere complexes comprising microspheres coupled to norovirus virus like particles.

What is claimed is: 
     
         1 . A microsphere complex comprising a microsphere coupled to a norovirus virus like particle (VLP). 
     
     
         2 . The microsphere complex of  claim 1 , wherein the norovirus VLP comprises the major viral capsid protein VP1 and optionally the minor viral capsid protein VP2. 
     
     
         3 . The microsphere complex of  claim 2 , wherein the major viral capsid protein VP1 is at least 80% or at least 85% or at least 90% or at least 95% or 100% identical to SEQ ID NO: 1 or SEQ ID NO: 2 or SEQ ID NO: 3 or SEQ ID NO: 4 or SEQ ID NO: 5 or SEQ ID NO: 6 or SEQ ID NO: 7 or SEQ ID NO: 8 or SEQ ID NO: 9 or SEQ ID NO: 10 or SEQ ID NO: 11 or SEQ ID NO: 12 or SEQ ID NO: 13 or SEQ ID NO: 14 or SEQ ID NO: 15 or SEQ ID NO: 16 or SEQ ID NO: 17 or SEQ ID NO: 18 or SEQ ID NO: 19 or SEQ ID NO: 20 or SEQ ID NO: 21 or SEQ ID NO: 22. 
     
     
         4 . The microsphere complex of any one of  claims 1 to 3 , wherein the microsphere comprises a detectable label. 
     
     
         5 . The microsphere complex of  claim 4 , wherein the detectable label is at least one fluorescent dye. 
     
     
         6 . The microsphere complex of  claim 5 , wherein the microsphere can be identified by the emission signal of the at least one fluorescent dye upon irradiation with a light source. 
     
     
         7 . A kit comprising an amount of at least one microsphere complex of any one of  claims 1 to 6  and optionally an amount of a detection antibody. 
     
     
         8 . A kit comprising:
 an amount of at least one microsphere complex of any one of  claims 1 to 6 , and   an amount of at least one reporter antibody that binds to the norovirus VLP of the at least one microsphere complex.   
     
     
         9 . The kit according to  claim 8 , wherein the at least one reporter antibody is attached to a detectable label by the heavy chain constant region of the at least one reporter antibody. 
     
     
         10 . The kit of  claim 9 , wherein the at least one reporter antibody is indirectly attached to the detectable label by the heavy chain constant region of the at least one reporter antibody, wherein the reporter antibody reacts with a secondary reporter antibody directly attached to a detectable label. 
     
     
         11 . The kit of  claims 9 and 10 , wherein the detectable label is a fluorescence label, preferably wherein the fluorescence label is selected from the group consisting of xanthene, fluorescein isothiocyanate, rhodamine, phycoerythrin, cyanine, coumarin, and any derivative thereof. 
     
     
         12 . The kit according to any one of  claims 8 to 11 , wherein the at least one reporter antibody provides an EC 50  value towards the norovirus VLP of the at least one microsphere complex of less than 0.5 μg/mL, or less than 0.4 μg/mL or less than 0.3 μg/mL or less than 0.2 μg/mL or less than 0.15 μg/mL or less than 0.1 μg/mL or less than 0.09 μg/mL or less than 0.08 μg/mL or less than 0.07 μg/mL or less than 0.05 μg/mL or less than 0.03 μg/mL or less than 0.02 μg/mL or less than 0.01 μg/mL. 
     
     
         13 . The kit according to any one of  claims 8 to 12 , wherein the at least one reporter antibody comprises
 a heavy chain variable region (VH) amino acid sequence as represented by SEQ ID NO: 27, and   a light chain variable region (VL) amino acid sequence as represented by SEQ ID NO: 28.   
     
     
         14 . The kit according to any one of  claims 8 to 12 , wherein the at least one reporter antibody comprises
 a heavy chain variable region (VH) amino acid sequence as represented by SEQ ID NO: 29, and   a light chain variable region (VL) amino acid sequence as represented by SEQ ID NO: 30.   
     
     
         15 . The kit according to any one of  claims 8 to 12 , wherein the kit comprises
 an amount of two microsphere complexes according to any one of  claims 1 to 6 ,
 wherein the first microsphere complex comprises a first microsphere coupled to a first norovirus VLP and the second microsphere complex comprises a second microsphere coupled to a second norovirus VLP, and 
 wherein the first microsphere comprises a first detectable label and the second microsphere comprises a second detectable label and wherein the emission signal of the first detectable label differs from the emission signal of the second detectable label, and 
   and an amount of two reporter antibodies,
 wherein the first reporter antibody binds to the first norovirus VLP and does not bind to the second norovirus virus like particle, and 
 wherein the second reporter antibody binds to the second norovirus VLP and does not bind to the first norovirus VLP. 
   
     
     
         16 . The kit according to  claim 15 , wherein the first or the second reporter antibody comprises
 a heavy chain variable region (VH) amino acid sequence as represented by SEQ ID NO: 27, and   a light chain variable region (VL) amino acid sequence as represented by SEQ ID NO: 28;   or   a heavy chain variable region (VH) amino acid sequence as represented by SEQ ID NO: 29, and   a light chain variable region (VL) amino acid sequence as represented by SEQ ID NO: 30.   
     
     
         17 . The kit according to  claim 15 , wherein the first norovirus VLP is a GI.1 VLP and the second norovirus virus like particle is a GII.4/Consensus VLP. 
     
     
         18 . The kit according to  claim 17 , wherein the second reporter antibody comprises
 a heavy chain variable region (VH) amino acid sequence as represented by SEQ ID NO: 29, and   a light chain variable region (VL) amino acid sequence as represented by SEQ ID NO: 30;   and optionally wherein the first reporter antibody comprises   a heavy chain variable region (VH) amino acid sequence as represented by SEQ ID NO: 27, and   a light chain variable region (VL) amino acid sequence as represented by SEQ ID NO: 28.   
     
     
         19 . The kit according to any one of  claims 8 to 14 , wherein the kit comprises an amount of one microsphere complex according to any one of  claims 1 to 6  and an amount of one reporter antibody that binds to the norovirus VLP of the microsphere complex. 
     
     
         20 . A method for detecting a signal from a detection antibody indicative for the presence and/or amount of norovirus-reactive antibodies in a sample from a subject comprising the steps of:
 Step 1: contacting an amount of a microsphere complex according to any one of  claims 1 to 6  with the sample to allow binding of the norovirus-reactive antibodies in the sample to the norovirus virus like particles (VLPs) coupled to the microspheres in the microsphere complex,   Step 2: contacting an amount of a detection antibody with the norovirus-reactive antibodies bound to the norovirus VLPs in step 1 to allow binding of the detection antibody to the heavy chain constant region of the norovirus-reactive antibodies, wherein the detection antibody binds to the norovirus-reactive antibodies with the variable region of the detection antibody and wherein the detection antibody is attached to a detectable label, and   Step 3: detecting a signal from the detection antibody bound to the norovirus-reactive antibodies in step 2.   
     
     
         21 . The method according to  claim 20  for determining the presence and/or amount of norovirus-reactive antibodies in a sample from a subject, wherein the method comprises the further steps of:
 Step 4: determining the presence and/or amount of the detection antibody bound to the norovirus-reactive antibodies from the signal of step 3, and 
 Step 5: determining the presence and/or amount of norovirus-reactive antibodies in the sample from the presence and/or amount of the detection antibody determined in step 4. 
 
     
     
         22 . A method for detecting a signal from a detection antibody indicative for the presence and/or amount of norovirus-reactive antibodies in a sample from a subject comprising the steps of:
 Step 1: contacting an amount of at least two microsphere complexes according to any one of  claims 1 to 6 ,
 wherein the first microsphere complex comprises a first microsphere coupled to a first norovirus VLP and the second microsphere complex comprises a second microsphere coupled to a second norovirus VLP, and 
 wherein the first microsphere comprises a first detectable label and the second microsphere comprises a second detectable label and wherein the emission signal of the first detectable label differs from the emission signal of the second detectable label, 
   with the sample to allow binding of the norovirus-reactive antibodies in the sample to the first and/or the second norovirus VLP,   Step 2: contacting an amount of a detection antibody with the norovirus-reactive antibodies bound to the first and/or the second norovirus VLP in step 1 to allow binding of the detection antibody to the heavy chain constant region of the norovirus-reactive antibodies, wherein the detection antibody binds to the norovirus-reactive antibodies with the variable region of the detection antibody and wherein the detection antibody is attached to a third detectable label,   Step 3: detecting the emission signal of the detectable label of at least one microsphere upon irradiation with a first light source and comparing the emission signal with the emission signal of the first detectable label and with the emission signal of the second detectable label, thereby identifying the at least one microsphere and the norovirus VLP the at least one microsphere is coupled to, and   simultaneously detecting a signal from the detection antibody bound to the norovirus-reactive antibodies bound to the norovirus VLP of the at least one microsphere in step 2 upon irradiation with a second light source,   Step 4: repeating step 3 until at least 30 microspheres coupled to the same norovirus VLP are identified, and   Step 5: summarizing the detected signal from the detection antibody in step 3 for all identified microspheres coupled to the same norovirus VLP, wherein the summarized signal is indicative for the presence and/or amount of norovirus-reactive antibodies in the sample.   
     
     
         23 . The method according to  claim 22  for determining the presence and/or amount of norovirus-reactive antibodies in a sample from a subject, wherein the method comprises the further steps of:
 Step 6: determining the presence and/or amount of the detection antibody bound to the norovirus-reactive antibodies from the summarized signal of step 5, and 
 Step 7: determining the presence and/or amount of norovirus-reactive antibodies in the sample from the presence and/or amount of the detection antibody determined in step 6. 
 
     
     
         24 . The method of  claim 22 or 23 , wherein in step 1 an amount of at least five or at least ten or at least fifteen or at least twenty microsphere complexes is contacted with the sample. 
     
     
         25 . The method of any one of  claims 22 to 24 , wherein in step 1 an amount of a first microsphere complex comprising a first microsphere coupled to a GI.1 VLP, an amount of a second microsphere complex comprising a second microsphere coupled to a GI.2 VLP, an amount of a third microsphere complex comprising a third microsphere coupled to a GI.3 VLP, an amount of a fourth microsphere complex comprising a fourth microsphere coupled to GI.4 VLP, an amount of a fifth microsphere complex comprising a fifth microsphere coupled to a GI.5 VLP, an amount of a sixth microsphere complex comprising a sixth microsphere coupled to a GI.6 VLP, an amount of a seventh microsphere complex comprising a seventh microsphere coupled to a GI.7 VLP, an amount of an eight microsphere complex comprising an eight microsphere coupled to GII.1 VLP, an amount of a ninth microsphere complex comprising a ninth microsphere coupled to a GII.2 VLP, an amount of a tenth microsphere complex comprising a tenth microsphere coupled to a GII.3 VLP, an amount of an eleventh microsphere complex comprising an eleventh microsphere coupled to a GII.4/Consensus VLP, an amount of a twelfth microsphere complex comprising a twelfth microsphere coupled to GII.4/Sydney VLP, an amount of a thirteenth microsphere complex comprising a thirteenth microsphere coupled to a GII.4/New Orleans VLP, an amount of a fourteenth microsphere complex comprising a fourteenth microsphere coupled to a GII.4/Yerseke VLP, an amount of a fifteenth microsphere complex comprising a fifteenth microsphere coupled to a GII.4/Den Haag VLP, an amount of a sixteenth microsphere complex comprising a sixteenth microsphere coupled to GII.6 VLP, an amount of a seventeenth microsphere complex comprising a seventeenth microsphere coupled to a GII.7 VLP, an amount of an eighteenth microsphere complex comprising an eighteenth microsphere coupled to a GII.12 VLP, an amount of a nineteenth microsphere complex comprising a nineteenth microsphere coupled to a GII.17/1978 VLP, and an amount of a twentieth microsphere complex comprising a twentieth microsphere coupled to GII.17/2015 VLP is contacted with the sample. 
     
     
         26 . The method according to any one of  claims 20 to 25 , wherein the detection antibody is directly attached to the detectable label by the heavy chain constant region of the detection antibody. 
     
     
         27 . The method according to any one of  claims 20 to 26 , wherein the detectable label the detection antibody is attached to is a fluorescence label, preferably wherein the fluorescence label is selected from the group consisting of xanthene, fluorescein isothiocyanate, rhodamine, phycoerythrin, cyanine, coumarin, and any derivative thereof. 
     
     
         28 . A method for detecting a signal from a reporter antibody indicative for the presence and/or amount of norovirus-reactive antibodies in a sample from a subject comprising the steps of:
 Step 1: providing a kit according to  claim 19 , including an amount of a microsphere complex and an amount of a reporter antibody,   Step 2: contacting the amount of the microsphere complex and the amount of the reporter antibody with the sample to allow binding of the norovirus-reactive antibodies in the sample to the norovirus VLPs coupled to the microspheres in the microsphere complex while competing with the reporter antibody, and   Step 3: detecting a signal from the reporter antibody bound to the norovirus VLPs in step 2.   
     
     
         29 . The method according to  claim 28 , comprising the steps of:
 Step 1: providing a kit according to  claim 19 , including an amount of a microsphere complex and an amount of a reporter antibody,   Step 2.1: contacting the amount of the microsphere complex of step 1 with the sample to allow binding of norovirus-reactive antibodies in the sample to the norovirus VLPs coupled to the microspheres in the microsphere complex,   Step 2.2: contacting the amount of the reporter antibody with the microsphere complex and the sample of step 2.1 to allow binding of the reporter antibody to the norovirus VLPs coupled to the microspheres in the microsphere complex, and   Step 3: detecting a signal from the reporter antibody bound to the norovirus VLPs in step 2.2.   
     
     
         30 . The method according to  claim 28 , comprising the steps of:
 Step 1: providing a kit according to  claim 19 , including an amount of a microsphere complex and an amount of a reporter antibody,   Step 2.1: contacting the amount of the microsphere complex of step 1 with the sample to allow binding of norovirus-reactive antibodies in the sample to the norovirus VLPs coupled to the microspheres in the microsphere complex,   Step 2.2: contacting the amount of the reporter antibody with the microsphere complex and the sample of step 2.1 to allow binding of the reporter antibody to the norovirus VLPs coupled to the microspheres in the microsphere complex,   Step 2.3: contacting the amount of reporter antibody, the amount of microsphere complex, and the sample of step 2.2 with an amount of a secondary reporter antibody to allow binding of the secondary reporter antibody to the constant region of the reporter antibody, and   Step 3: detecting a signal from the secondary reporter antibody bound to the reporter antibody in step 2.3, wherein the reporter antibody is bound to the norovirus VLPs in step 2.2.   
     
     
         31 . The method according to any one of  claims 28 to 30  for determining the presence and/or amount of norovirus-reactive antibodies in a sample from a subject, wherein the method comprises the further steps of:
 Step 4: determining the presence and/or amount of the reporter antibody from the signal of step 3, and 
 Step 5: determining the presence and/or amount of norovirus-reactive antibodies in the sample from the presence and/or amount of the reporter antibody determined in step 4. 
 
     
     
         32 . A method for detecting a signal from a reporter antibody indicative for the presence and/or amount of norovirus-reactive antibodies in a sample from a subject comprising the steps of:
 Step 1: providing a kit according to any one of  claims 8 to 18 , including an amount of at least two microsphere complexes,
 wherein the first microsphere complex comprises a first microsphere coupled to a first norovirus VLP and the second microsphere complex comprises a second microsphere coupled to a second norovirus VLP, and 
 wherein the first microsphere comprises a first detectable label and the second microsphere comprises a second detectable label and wherein the emission signal of the first detectable label differs from the emission signal of the second detectable label, and 
   and an amount of at least two reporter antibodies,
 wherein the first reporter antibody binds to the first norovirus VLP and does not bind to the second norovirus virus like particle, and 
 wherein the second reporter antibody binds to the second norovirus VLP and does not bind to the first norovirus VLP; 
   Step 2: contacting the amount of the at least two microsphere complexes with the sample to allow binding of the norovirus-reactive antibodies in the sample to the first and/or the second norovirus VLPs while competing with the at least two reporter antibodies;   Step 3: detecting the emission signal of the detectable label of at least one microsphere upon irradiation with a first light source and comparing the emission signal with the emission signal of the first detectable label and with the emission signal of the second detectable label, thereby identifying the at least one microsphere and the norovirus VLP the at least one microsphere is coupled to, and   simultaneously detecting a signal from the reporter antibody bound to the norovirus VLPs of the at least one microsphere in step 2 upon irradiation with a second light source;   Step 4: repeating step 3 until at least 30 microspheres coupled to the same norovirus VLP are identified; and   Step 5: summarizing the detected signal from the reporter antibody in step 3 for all identified microspheres coupled to the same norovirus VLP, wherein the summarized signal is indicative for the presence and/or amount of norovirus-reactive antibodies in the sample.   
     
     
         33 . The method of  claim 32 , comprising the steps of:
 Step 1: providing a kit according to any one of  claims 8 to 18 , including an amount of at least two microsphere complexes,
 wherein the first microsphere complex comprises a first microsphere coupled to a first norovirus VLP and the second microsphere complex comprises a second microsphere coupled to a second norovirus VLP, and 
 wherein the first microsphere comprises a first detectable label and the second microsphere comprises a second detectable label and wherein the emission signal of the first detectable label differs from the emission signal of the second detectable label, and 
   and an amount of at least two reporter antibodies,
 wherein the first reporter antibody binds to the first norovirus VLP and does not bind to the second norovirus virus like particle, and 
 wherein the second reporter antibody binds to the second norovirus VLP and does not bind to the first norovirus VLP; 
   Step 2.1: contacting the amount of the at least two microsphere complexes with the sample to allow binding of the norovirus-reactive antibodies in the sample to the first and/or the second norovirus VLPs;   Step 2.2: contacting the amount of the at least two reporter antibodies with the at least two microsphere complexes and the sample of step 2.1 to allow binding of the at least two reporter antibodies to the norovirus VLPs coupled to the microspheres;   Step 3: detecting the emission signal of the detectable label of at least one microsphere upon irradiation with a first light source and comparing the emission signal with the emission signal of the first detectable label and with the emission signal of the second detectable label, thereby identifying the at least one microsphere and the norovirus VLP the at least one microsphere is coupled to, and   simultaneously detecting a signal from the reporter antibody bound to the norovirus VLPs of the at least one microsphere in step 2.2 upon irradiation with a second light source;   Step 4: repeating step 3 until at least 30 microspheres coupled to the same norovirus VLP are identified; and   Step 5: summarizing the detected signal from the reporter antibody in step 3 for all identified microspheres coupled to the same norovirus VLP, wherein the summarized signal is indicative for the presence and/or amount of norovirus-reactive antibodies in the sample.   
     
     
         34 . The method of  claim 32 , comprising the steps of:
 Step 1: providing a kit according to any one of  claims 8 to 18 , including an amount of at least two microsphere complexes,
 wherein the first microsphere complex comprises a first microsphere coupled to a first norovirus VLP and the second microsphere complex comprises a second microsphere coupled to a second norovirus VLP, and 
 wherein the first microsphere comprises a first detectable label and the second microsphere comprises a second detectable label and wherein the emission signal of the first detectable label differs from the emission signal of the second detectable label, and 
   and an amount of at least two reporter antibodies,
 wherein the first reporter antibody binds to the first norovirus VLP and does not bind to the second norovirus virus like particle, and 
 wherein the second reporter antibody binds to the second norovirus VLP and does not bind to the first norovirus VLP; 
   Step 2.1: contacting the amount of the at least two microsphere complexes with the sample to allow binding of the norovirus-reactive antibodies in the sample to the first and/or the second norovirus VLPs;   Step 2.2: contacting the amount of the at least two reporter antibodies with the at least two microsphere complexes and the sample of step 2.1 to allow binding of the at least two reporter antibodies to the norovirus VLPs coupled to the microspheres;   Step 2.3: contacting the amount of the at least two reporter antibodies, the amount of the at least two microsphere complexes and the sample of step 2.2 with an amount of a secondary reporter antibody to allow binding of the secondary reporter antibody to the constant region of the at least two reporter antibodies;   Step 3: detecting the emission signal of the detectable label of at least one microsphere upon irradiation with a first light source and comparing the emission signal with the emission signal of the first detectable label and with the emission signal of the second detectable label, thereby identifying the at least one microsphere and the norovirus VLP the at least one microsphere is coupled to, and   simultaneously detecting a signal from the secondary reporter antibody bound to the reporter antibody bound to the norovirus VLPs of the at least one microsphere in step 2.3 upon irradiation with a second light source;   Step 4: repeating step 3 until at least 30 microspheres coupled to the same norovirus VLP are identified; and   Step 5: summarizing the detected signal from the secondary reporter antibody in step 3 for all identified microspheres coupled to the same norovirus VLP, wherein the summarized signal is indicative for the presence and/or amount of norovirus-reactive antibodies in the sample.   
     
     
         35 . The method according to any one of  claims 32 to 34 , wherein the method comprises the further steps of:
 Step 6: determining the presence and/or amount of the reporter antibody from the summarized signal of step 5, and   Step 7: determining the presence and/or amount of norovirus-reactive antibodies in the sample from the presence and/or amount of the reporter antibody determined in step 6.   
     
     
         36 . The method according to any one of  claims 32 to 35 , wherein the kit in step 1 provides an amount of two microsphere complexes and an amount of two reporter antibodies. 
     
     
         37 . The method of  claim 36 , wherein the first microsphere complex comprises a first microsphere coupled to a GI.1 VLP and wherein the second microsphere complex comprises a second microsphere coupled to a GII.4/Consensus VLP. 
     
     
         38 . A method for diagnosing a norovirus infection in a subject comprising the steps of:
 Step 1: providing a sample from the subject outside the subject body,   Step 2: determining the amount of norovirus-reactive antibodies in the sample according to any one of claims  20  to  37 , and   Step 3: determining infection by comparing the amount of norovirus-reactive antibodies to established amounts of norovirus-reactive antibodies in norovirus infected subjects.   
     
     
         39 . A method for determining protection of a subject against a norovirus infection comprising the steps of:
 Step 1: providing a sample from the subject outside the subject body,   Step 2: determining the amount of norovirus-reactive antibodies in the sample according to any one of claims  20  to  37 , and   Step 3: determining protection by comparing the amount of norovirus-reactive antibodies in step 2 to protective amounts of norovirus-reactive antibodies.   
     
     
         40 . The method according to any one of  claims 20 to 39 , wherein the subject is a human.